MMCF enables a highly stable microbial coculture
We first aimed to establish a stable coculture system whose population composition is insensitive to the IIRs and can converge onto a narrow range, which would greatly reduce the production fluctuation and facilitate scale-up. We used the prokaryotic model organism E. coli to construct a homogenous coculture and step-wise improved the stability by enhancing the correlation between the strains (Fig. 1). E. coli BW25113 and BW25113ΔpykAΔpykF were used as the basal strains. To reduce competition, two derivatives were generated to use glycerol and glucose, respectively. In E. coli, glucose is converted to glucose-6-phosphate via the phosphotransferase system (PTS) or glucokinase35. To block glucose catabolism, the relevant genes ptsG (encoding the glucose-specific transporter of the PTS), manXYZ (encoding the mannose PTS that can also transport glucose), and glk (encoding glucokinase) in BW25113ΔpykAΔpykF were deleted, generating the glycerol-utilizing strain Bgly1. Noteworthy, with the deletion of the two pyruvate kinase genes (pykA and pykF), the carbon flux would enter the TCA cycle primarily via the phosphoenolpyruvate carboxylase (encoded by ppc) by-pass. To block glycerol catabolism, gene glpK (encoding glycerol kinase) in BW25113 was deleted, generating the glucose-utilizing strain Bglc1. The glycerol dehydrogenase and dihydroxyacetone kinase-mediated glycerol dissimilation pathway was kept intact because it works mainly under anaerobic conditions36. Because strain Bgly1 and Bglc1 use separate carbon sources for growth and do not require essential ingredients from each other, their coculture is regarded as neutralistic.
We determined the growth profiles of Bgly1 and Bglc1 monocultures. The two strains showed specific utilization of glycerol and glucose, respectively. Bgly1 exhibited an exponential growth phase till 20 h in glycerol minimal media while Bglc1 quickly reached the stationary phase at 10 h in glucose minimal media (Supplementary Fig. 1). The two strains were transformed respectively with plasmids pSA-eGFP and pSA-mcherry and were cocultured at different IIRs (80%, 50%, and 20%, i.e., 4:1, 1:1, and 1:4; the same below). Cell growth was monitored by spectrometry and the population was determined by flow cytometry. At the population level, the apparent growth profile of the coculture is similar to the Bglc1 monoculture, and would reach the stationary phase in 12 h; the total cell density (measured by OD600) was positively related to the IIRs of Bgly1 (Fig. 2a). At the strain level, Bglc1 dominated in the coculture, and its final percentage at 48 h ranged from 93.7 to 82.2% at the three IIRs (Fig. 2a). Both cell growth and population composition are affected by the IIRs, indicating the necessity to further improve the coculture stability.
a The Neutralistic coculture; b the Commensalistic coculture; c the Mutualistic coculture. IIRs, the initial inoculation ratios. Data shown are mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.
To this end, a commensalistic coculture was constructed based on the above neutralistic coculture. Amino acids are frequently used as cross-feeding metabolites, and the biosynthetically costly ones such as methionine, lysine, and aromatics were shown to promote stronger correlation27. However, the single-metabolite correlation is relatively loose and insufficient to maintain stability under changing culture conditions. Further, many amino acid pairs are unable to sustain the normal growth of the cocultures due to their low leak into the extracellular environment27. Instead, we created a multi-metabolite correlation by engineering the glutamate node. Glutamate plays a central role in amino acid metabolism, and is the common amino doner for amino acid biosynthesis. E. coli synthesizes glutamate from ammonia via two pathways catalyzed by glutamate dehydrogenase (encoded by gdhA) and glutamate synthase (encoded by gltBD), respectively. As these genes were deleted in strain Bglc2, it was unable to grow in glucose minimal media. Supplement of glutamate recovered growth in a dose-dependent manner, and supplement of other amino acids such as phenylalanine and tyrosine partially recovered cell growth via the endogenous transamination (Supplementary Fig. 2a and 2b). With this design, the amino acid metabolic networks of strains Bgly1 and Bglc2 would be connected, forming multi-point interactions. Because Bglc2 relies on amino acids from Bgly1 for growth while Bgly1 can grow independently, the Bgly1/Bglc2 coculture is regarded as commensalistic.
Encouragingly, we noticed that the total cell density of the Bgly1/Bglc2 coculture converged to around 7 at the three different IIRs (Fig. 2b). Bgly1 becomes the dominant strain, indicating that the growth of Bglc2 is limited by the supply of amino acids from Bgly1. Although the final ratios of Bgly1 tended to converge, they still ranged from 74.8 to 87.8% at 48 h (Fig. 2b). The results indicate that the stability of the commensalistic coculture is not significantly improved compared with the neutralistic coculture, likely because strain Bgly1 is lack of restriction.
To strengthen the correlation between the members, we further constructed a mutualism coculture. Besides amino acid anabolism, energy metabolism is another essential function to support cell growth. The TCA cycle, coupling with the oxidative respiratory chain, is the major energy source under aerobic conditions. In addition, it contains 3 out of the 12 precursor metabolites that connect cell catabolism and anabolism (oxaloacetate, α-ketoglutarate, succinyl-CoA), and the involved carboxylic acids can transfer across cell membranes. Thus, the TCA cycle was selected as a second cross-feeding branch. As pykA and pykF were already deleted in Bgly1, gene ppc was further deleted to block the carbon flux to the TCA cycle, yielding strain Bgly2. As expected, Bgly2 was unable to grow in glycerol minimal media, and the growth can only be fully covered by co-addition of multiple carboxylic acids (Supplementary Fig. 3).
In the Bgly2/Bglc2 coculture, Bgly2 restored growth, confirming that Bglc2 can provide the TCA cycle metabolites. With such a multi-metabolite cross-feeding design, not only the total cell growth but also the population composition converged over time, and the percentages of strain Bgly2 were stabilized at around 76% in 48 h (Fig. 2c). Taken together, we can see that establishing a close multi-metabolite mutualistic relationship between the strains is essential to constructing a stable coculture system.
To determine the metabolites involved in cross-feeding, we performed the extracellular metabolomic analysis of the neutralistic and the mutualism cocultures. A total of 5 TCA cycle intermediates and 9 amino acids were detected in the supernatants of both cocultures, among which the levels of α-ketoglutarate and glutamate were significantly higher in the mutualistic coculture than in the neutralistic coculture (Supplementary Fig. 4). Considering the essential roles of α-ketoglutarate and glutamate in the TCA cycle and amino acid metabolism, we infer that they may serve as the key metabolites for crossing-feeding while the other relevant metabolites (carboxylic acids in the TCA cycle and amino acids) detected and even non-detected can also contribute to the cross-feeding.
The stable culture system enables efficient salidroside biosynthesis
To investigate the applicability in actual production, we compared the three coculture systems (neutralistic, commensalistic, and mutualistic) for heterologous chemical biosynthesis. Salidroside, an active plant natural product, was previously used as a demonstration of an E. coli single cross-feeding coculture system. For better comparison, we chose it as the first target compound. The aglycone tyrosol is synthesized via the shikimate pathway with tyrosine as a precursor, and is further glycosylated to salidroside with UDP-glucose as a glucosyl donor (Supplementary Fig. 5). Accordingly, the pathway was divided and allocated to the members of the cocultures, where the glycerol-utilizing strains are responsible for the upstream tyrosine biosynthesis while the glucose-utilizing strains for the downstream conversion of tyrosine to salidroside (Fig. 3a).
a Schematic of the E. coli-E. coli cocultures to accommodate the salidroside biosynthetic pathway and convert a glycerol and glucose mixture to salidroside (The salidroside biosynthetic pathway is shown in orange. For the detailed pathway, see Supplementary Fig. 5). Curves of cell growth and population change in b the Neutralistic Bgly1-Tyr/Bglc1-Sal coculture, c the Commensalistic Bgly1-Tyr/Bglc2-Sal coculture, and d the Mutualistic Bgly2-Tyr/Bglc2-Sal coculture. e Comparison of salidroside titers in the three coculture systems. f Scale-up production of salidroside using the Bgly2-Tyr/Bglc2-Sal coculture. MMCF multi-metabolite cross-feeding, IIRs the initial inoculation ratios. Metabolites: PEP phosphoenolpyruvate, G6P glucose-6-phosphate, UDPG uridine diphosphate glucose, α-KG α-ketoglutarate, L-GLU L-glutamate, AAs amino acids. Data shown are mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.
The relative stability of the cocultures was maintained after loading with the biosynthetic pathway, and the mutualistic coculture Bgly2-Tyr/Bglc2-Sal was the most stable with different IIRs. In the neutralistic coculture, the upstream strain Bgly1-Tyr took a minor percentage (10.5–21.4%), which limits the precursor supply and so does the final salidroside titers (337–683 mg/L) (Fig. 3b, e). In the commensalistic coculture, Bgly1-Tyr dominated throughout the cultivation process and its final ratios ranged from 89.5 to 73.2% (Fig. 3c); the salidroside titers ranged from 759 to 1276 mg/L at 48 h. (Fig. 3e). In the mutualistic coculture, the total cell growth, population composition, and salidroside titers were all strikingly stable with different IIRs. Although the IIRs varied from 4:1 to 1:4, the average salidroside titers at 48 h were stabilized at 1552 ± 27 mg/L, and the final percentages of Bgly2-Tyr converged at around 75% at 48 h (Fig. 3d and e). Interestingly, the amount of glycerol consumed (4.11 g/L at 48 h) was much less than that of glucose (9.69 g/L at 48 h) (Supplementary Fig. 6a), indicating that a significant amount of carbon flux derived from glucose is transferred from Bglu2-Sil to Bgly2-Tyr.
To explore the long-term stability of the mutualistic coculture, we conducted continuous passage cultivation in shake flasks. Every 24 h, the cell culture was taken as the seed and inoculated into the next fresh medium. The result showed that the coculture can maintain good production and population stability for up to 10 days (Supplementary Fig. 7). Interestingly, the titer even showed a gradual increase with the passage subcultures, indicating that the coordination between the counterparts may be further improved during the cultivation.
One feature of artificial microbial consortia is the division of labor. To investigate whether the mantainance of two populations causes the loss of production yield, we compared the mutralistic coculture with the monoculture for salidroside production. The result showed that the yield by the coculture (0.11 g/g) is equal to that by the monoculture, suggesting that the coculture does not necessarily sacrifice the carbon yield (Supplementary Fig. 6b). The slightly lower titer by the coculture (1523 mg/L versus 1603 mg/L) is attributed to the lower cell density (7.35 versus 8.56 at 48 h) (Supplementary Fig. 6c and d).
To evaluate the scalability of the mutualistic coculture, salidroside production was performed in 3 l bioreactors at a IIR of 50%. The percentage of strain Bgly2-Tyr declined rapidly to 30.3% during the first 12 h, then recovered to 73.2% at 24 h, maintained stable till 48 h, and finally slightly declined to 68.9% at 84 h (Fig. 3f). The general stability was maintained during the cultivation period. The initial perturbation should be attributed to that the nutritions in the seed culture (yeast extract and peptone) promote the growth of strain Bglc2. As for salidroside production, the titer increased continuously to 12.52 g/L at 84 h, with the yield of 0.12 g/g total carbon sources and the productivity of 0.15 g/L/h. The titer and productivity are doubled and tripled compared to those resulting in the previous study10, respectively. The results showed that the mutualistic coculture could tolerate disturbance and maintain stability at bioreactor level, paving the way for its industrial application.
Introducing a metabolite-responsive biosensor increases system tunability
Besides stability, a versatile coculture system should also possess tunability to autonomously balance metabolic fluxes between the partial pathways distributed in different strains. To show the advantage of tunability, we chose coniferol biosynthesis as the second example. Coniferol is also synthesized via the shikimate pathway37. Briefly, tyrosine is converted to caffeate by tyrosine ammonia lyase (TAL) and 4-hydroxyphenylacetate 3-hydroxylase (HpaBC), and caffeate is then converted to coniferol viasequential reactions catalyzed by p-coumarate-CoA ligase (4CL1), caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), and alcohol dehydrogenase (ADH) (Supplementary Fig. 5). The biosynthetic pathway was divided and introduced into strains Bgly2 and Bglc2, generating strains Bgly2-Caf and Bglc2-Con (Fig. 4a) In the Bgly2-Caf/Bglc2-Con coculture, the population percentages of strain Bglc2-Con ranged from 10 to 16.5% at 48 h (Fig. 4b). Coniferol was produced at low titers (62, 85, and 95 mg/L) with the accumulation of caffeate (175, 140, and 110 mg/L) at the three IIRs (Fig. 4c), indicating that the downstream strain Bglc2-Con is incapable of completing the conversion. Thus, it is necessary to modulate the strain population according to the actual demand of the pathway. As shown in Supplementary Fig. 2a and 2b, the population composition can be adjusted by supplementing ingredients such as glutamate or yeast extract. However, the doses need to be manually titrated and oftentimes is difficult to control precisely in large-scale production. Alternatively, the population composition can be modulated by fine-tuning the expression of gdhA in strain Bglc2-Con, but the static control using constitutive promoters may not match the dynamic change of cell metabolism during the cultivation process. Instead, we proposed to control gdhA expression with a caffeate-responsive biosensor, through which the accumulation of caffeate would therefore activate gdhA expression and promote the growth of strain Bglc2-Con. However, the implementation was hindered by the lack of known caffeate-responsive biosensors. A set of biosensors for aromatic compounds have been characterized38,39,40. According to their detection spectrum, the phenol-responsive DmpR biosensor and the salicylate-responsive NahR biosensor were inferred to be promising candidates. Their responsiveness was verified and assessed using a fluorescence reporter system. In brief, the transcriptional factors NahR and DmpR were constitutively expressed by the J23101 promoter, and the egfp gene was controlled by their cognate promoters. The DmpR biosensor showed expected responsiveness to caffeate with a 12-fold increase in fluorescence intensity at 2 mM caffeate than the control, while the NahR did not (Supplementary Fig. 8a and 8b). In comparison, 0.25 mM phenol led to a 50-fold increase (Supplementary Fig. 8c), indicating that caffeate has a lower affinity to DmpR than phenol. Besides, the DmpR biosensor showed only weak responsiveness (less than one-fold) to other pathway intermediates (L-dopa, tyrosine, and p-coumaric acid) (Supplementary Fig. 8d).
a Schematic of the E. coli-E. coli cocultures to accommodate the coniferol biosynthetic pathway and convert a glycerol and glucose mixture to coniferol (The coniferol biosynthetic pathway is shown in sapphire. For the detailed pathway, see Supplementary Fig. 5). b Curves of cell growth and population change in the static Bgly2-Caf/Bglc2-Con coculture. c Titers of coniferol and caffeate at 48 h in the Bgly2-Caf/Bglc2-Con coculture. d Curves of cell growth and population change in the dynamic Bgly2-Caf/Bglc2-ConDmpR coculture. e Titers of coniferol and caffeate at 48 h in the Bgly2-Caf/Bglc2-ConDmpR coculture. MMCF multi-metabolite cross-feeding, IIRs the initial inoculation ratios. Genes: gdhA encodes glutamate dehydrogenase, gltBD encodes glutamate synthase, Enzymes: GdhA glutamate dehydrogenase, DmpR the caffeate-responsive transcription factor. Metabolites: PEP phosphoenolpyruvate, α-KG α-ketoglutarate, L-GLU L-glutamate, AAs amino acids. Data shown are mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.
Considering the suitable sensitivity and specificity, the DmpR biosensor was used to control gdhA expression. GdhA was fused with a proteinase-degradation-tag SsrA to timely shutdown the response when the triggering signal disappears41,42. Strain Bglc2-Con was transformed with plasmid pSA-Pdmp-GS-PJ23101-dmpR, generating strain Bglc2-ConDmpR. In the Bgly2-Caf/Bglc2-ConDmpR coculture, the percentage of the downstream strain was increased from 16 to 52%, and the average total cell density (OD600) also increased from 5.9 to 7.8 (Fig. 4d). Consequently, coniferol titers were significantly improved (217, 258, and 246 mg/L) when only a small amount of caffeate accumulated (20, 5, and 4 mg/L) at the three IIRs (Fig. 4e). The results demonstrated that the population composition could be self-regulated while the stability is still maintained.
Establishing a three-strain coculture improves the production of silybin/isosilybin
To further explore the application potential, we used the stable and tunable coculture system for de novo production of the structurally more complicated compounds silybin and isosilybin, which have been used as hepatoprotectives43,44. They are biosynthesized via a divergent-convergent pathway (Supplementary Fig. 5). Two precursors (coniferol and taxifolin) are both derived from caffeate and are condensed to form silybin/isosilybin catalyzed by the ascorbate peroxidase APX145. Starting from tyrosine, the pathway involves 10 enzymatic steps. A two-step enzymatic cascade has been constructed for efficient biotransformation of eugenol and taxifolin to silybin/isosilybin46. Recently, the production of silybin/isosilybin has been achieved by combining metabolic engineering approaches with enzymatic catalysis47. In brief, the two precursors were separately synthesized from glucose using two metabolically engineered S. cerevisiae strains. After purification they were further converted to silybin/isosilybin by APX1. However, their one-pot de novo biosynthesis has not yet been achieved, suggesting the great challenges in modulating the full pathway.
The modularity of coculture engineering can simplify the construction and optimization process. As for silybin/isosilybin biosynthesis, the above caffeate-producing strain Bgly2-Caf can be used directly, and thus the focus was on the integration of the downstream modules. To guide the pathway assembly and optimization, the conversion capacities of the two branches were first evaluated. Two plasmids pZE-CCF4 and pZE-CA4C were constructed and used for the feeding experiments. When fed with 500 mg/L caffeate, the corresponding strains produced 268 mg/L of coniferol and 8 mg/L of taxifolin in 48 h, respectively (Supplementary Fig. 9), indicating that the taxifolin branch is rate-limiting. In addition, the peroxidase APX1 requires H2O2 as the co-substrate, and the NADH oxidase Nox from Lactococcus lactis was co-expressed to enable endogenous supply of H2O248. Accordingly, these modules were accommodated on plasmids pZE-CA, pCS-Plac-CF4C-PJ23101-CA, pSA-Pdmp-gdhA-PJ23101-DN. The plasmids were co-transferred into strain Bglc2, generating strain Bglc2-Sil. The coculture of Bglc2-Caf/Bglc2-Sil produced 0.28 mg/L of silybin/isosilybin in 48 h with the accumulation of 101 mg/L caffeate and 9 mg/L coniferol (Fig. 5b and c). The accumulation of caffeate did not further stimulate the growth of Bglc2-Sil, probably due to its heavy metabolic load (Fig. 5d).
a Schematic of the three-strain coculture to accommodate the silybin/isosilybin biosynthetic pathway and convert a glycerol and glucose mixture to silybin/isosilybin (The silybin/isosilybin biosynthetic pathway is shown in blue. For the detailed pathway, see Supplementary Fig. 5). b Silybin/isosilybin titers. c Amounts of the intermediates accumulated. d Population change of the two-strain coculture. e Population change of the three-strain coculture. MMCF multi-metabolite cross-feeding. Genes: gdhA encodes glutamate dehydrogenase, gltBD encodes glutamate synthase. Enzymes: GdhA glutamate dehydrogenase, DmpR the caffeate-responsive transcription factor. Metabolites: PEP phosphoenolpyruvate, α-KG α-ketoglutarate, L-GLU L-glutamate, AAs amino acids. Data shown are mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.
To reduce the metabolic burden and competition, the downstream pathway was distributed into two Bglc2 cells. The two strains Bglc2-Con(b) and Bglc2-Sil(b)were cocultured with the upstream strain Bglc2-Caf, forming a three-strain coculture (Fig. 5a). Silybin/isosilybin titers increased by six fold to 2.02 mg/L, and caffeate accumulation was reduced to 71 mg/L (Fig. 5b, c). In addition, the percentage of Bglc2-Sil(b) kept increasing to 72% within 48 h (Fig. 5e), showing the effectiveness of the biosensor. On the other hand, the percentage of Bglc2-Caf remained stable at around 18% after 12 h while that of Bglc2-Con(b) (without the biosensor) began to decrease after 24 h and dropped to 9% at 48 h (Fig. 5e). Successful biosynthesis of silybin/isosilybin demonstrates the potential of the dynamic mutralistic coculture for biosynthesis of complex chemicals.
